Subash_2025_Mol.Pharm__

Clopidogrel, a frequently used prodrug, is converted to its active metabolite through the intermediate 2-oxo-clopidogrel by cytochrome P450 (CYP) enzymes, which accounts for only 5%-15% of its metabolism. Majority of the clopidogrel dose (85%-90%) is extensively hydrolyzed to its inactive metabolite, clopidogrel carboxylic acid by carboxylesterase 1 (CES1). In vitro studies suggest the involvement of multiple CYP isoforms, with CYP1A2, CYP2C19, and CYP2B6 identified as major contributors to 2-oxo-clopidogrel formation. While CYP2C19 genetic polymorphisms are often highlighted as the primary factor contributing to variability in the clopidogrel response, the confounding role of CES1 variability on clopidogrel oxidation is less well understood. Our study utilizing proteomics-informed scaling highlights the importance of accurate estimation of the fraction metabolized (f(m)) by CES1 and CYPs in clopidogrel metabolism. The results also indicate that differential subcellular localization of these enzymes and technical variability in sample preparation can influence f(m) estimation, suggesting that HLM may not be an ideal model for investigating dual substrates of CYPs and CES. Quantitative proteomics and activity assays revealed significant variability in the absolute content and activities of CES1 and CYP enzymes across HLM donors (n = 10), which affected the estimation of f(mCES) versus f(mCYP). Human hepatocyte assay, which represents a CYP versus CES abundance ratio similar to that in liver tissue, demonstrated the critical roles of CYP3A4 and CES1 abundance in the 2-oxo-clopidogrel formation rate. Further, enzyme kinetic studies identified CYP3A4 as the primary contributor to 2-oxo-clopidogrel formation, but multiple other enzymes, including CYP2C9, were identified as contributors. Overall, our findings emphasize the need for accounting for variability in both CES1 and CYP enzymes to improve f(m) estimation in the in vitro to in vivo extrapolation of dual substrates of CYP/CES such as clopidogrel.